Elution mode, chromatography

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

Chromatographic separation of these mixtures in the elution mode is incapable of resolving many thousands of peptides present in these mixtures, even when orthogonal, two-dimensional separations are performed. The investigator is left with little option for low-abundance peptide iden-tihcation other than affinity approaches that target certain subclasses (e.g., phosphopeptides). While effective for certain applications, the latter allow for enrichment of only a small subset of low-abundance peptides. Because of its potential for broad applicability to the problem of low-abundance peptide enrichment, displacement chromatography remains a technique that offers great possibilities in this area. 

New concepts presented in this edition include monolithic columns, bonded stationary phases, micro-HPLC, two-dimensional comprehensive liquid chromatography, gradient elution mode, and capillary electromigration techniques. The book also discusses LC-MS interfaces, nonlinear chromatography, displacement chromatography of peptides and proteins, field-flow fractionation, retention models for ions, and polymer HPLC. 

Taylor and colleagues [98] at the Mayo Clinic published a method for the simultaneous analysis of urinary cortisol and cortisone. They used 2H4 cortisol as an internal standard and took a 0.5-ml urine sample. An API 2000 with Turboion-spray source was used in the positive-ion mode. Chromatography was conducted on a standard-bore C18 column with Q8 precolumn filter. MRM was conducted in the positive-ion mode monitoring m/z 363—>121 for cortisol, 367—>121 for 2TL, cortisol, and 361— -121 for cortisone. Cortisol and cortisone were separated and both were eluted within 2 min. Inter- and intra-assay variation for both compounds was < 9% for amounts above 2 pig/dl. The values obtained agree well with those of other studies, such as ours (Table 5.3.2) [62]. They found a range for cortisol for adult males of 4.2-60 pg/24 h and for adult females 3.0-43 pg/24 h. In summary, the 3-min run time of their method has allowed the Mayo group to completely transfer their cortisol and cortisone workload from RIA and HPLC to MS/MS. 

Hodges, R. S., Burke, T. W. L., and Mant, C. T. (1988). Preparative purification of peptides by reversed phase chromatography—sample displacement versus gradient elution modes. ]. Chromatogr. 444, 349-362. 

Displacement chromatography offers an attractive alternative to the elution mode of operation for preparative purification (S. Cramer, personal communication, 1999). Further investigations must be carried out to identify more cost-effective, nontoxic, and readily detectable displacers that are commercially available to the biotechnology industries. Displacers with high affinities in a range of commercially available stationary phases must be identified to facilitate the development of displacement steps on these materials. This will require significant advances in our understanding of the nature of affinity of these systems. 

Some other elution modes have been described. They are induced by various factors — cyclical field, secondary chemical equilibria, adhesion chromatography, asymmetrical electro-osmotic flow for a review, see Ref. 2. However, the number of their implementations is rather limited, and for this reason, these modes are not discussed here. 

Fig. 3 Chromatograms obtained by pH zone-refining CCC. (A) Separation of CBZ(Z) dipeptides. Experimental conditions are as follows apparatus type-J multilayer CPC (PC Inc., Potomac, MD, USA) with a 10-cm revolution radius column multilayer coil, 1.6-mm ID, 325 mL capacity solvent system methyl tert-h xiy ether/acetonitrile/water (2 2 3), 16 mM TEA in organic phase (pH 1.83), and 5.5 mM NH3 in aqueous phase (pH 10.62) sample eight CBZ(carbobenzyloxy) dipeptides as indicated in the chromatogram, each 100 mg in 50 mL of solvent (25 mL in each phase) flow rate 3.3 mL/hr in head-to-tail elution mode detection 206 nm revolution 800 rpm retention of stationary phase 65.1%. (B) Separation of bacitracin complex. High-performance liquid chromatography (HPLC) analysis indicated that two major components, bacitracins A and B, were isolated in peaks 3 and 5, respectively. Experimental conditions are as follows apparatus and column same as above solvent system methyl r -butyl ether/ acetonitrile/water (4 1 5), 40 mM triethylamine, 10% DEHPA in the organic stationary phase, and 20 mM HCl in aqueous mobile phase flow rate 3 mL/min sample 5 g of bacitracin dissolved in 40 mL of solvent (20 mL in each phase) revolution 800 rpm detection 280 nm.

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